Abstract Background Mitochondrial DNA (mtDNA) mutations are implicated in a wide range of diseases, underscoring the need to elucidate the relationship between mtDNA mutations and disease pathology. These diseases are often characterized by the presence of multiple mutations; however, research has been hampered by the lack of suitable animal models carrying multiplex mtDNA mutations. Such models cannot be produced through traditional breeding due to matrilineal inheritance of mtDNA. Methods Based on the TALE‐based mitochondrial genome editing tools, we generated rats harboring multiple mtDNA mutations by microinjecting mixed pairs of DdCBE plasmids into one‐cell‐stage zygotes. The efficiency of mtDNA editing and the potential off‐target effects were assessed through deep sequencing and long reads sequencing methodologies. Results In this study, we established double‐ and triple‐site mutant rats with the editing efficiencies of up to 58.5% and confirmed that these mutations can be stably transmitted through the germline. Moreover, our results demonstrated that DdCBE‐mediated mtDNA multi‐site editing of mtDNA exhibits minimal off‐target effects in both the mitochondrial and nuclear genomes in vivo. Conclusion This work represents the first successful generation of heritable multi‐site mtDNA mutant rats, providing a valuable model for elucidating the pathophysiological mechanisms of mitochondrial disorders and for developing potential therapeutics.

目的：目前线粒体碱基编辑器DdCBE仍然存在脱靶编辑较高的问题，拟通过优化DdCBE的结构来减少TALE序列依赖的脱靶，进一步提高编辑特异性。方法：对DdCBE中TALE结构的C端和N端氨基酸臂的长度和电荷进行改造以获得不同的编辑器变体；同时也通过引入脱氨酶抑制分子Dddi以抑制脱靶编辑。在线粒体基因组内源性位点开展测试，利用高通量测序的方式评估不同DdCBE变体的在靶编辑效率、脱靶编辑效率和编辑特异性。结果：DdCBE中TALE序列C端长度为41个氨基酸（C41），编辑特异性更高；替换TALE序列C端中的赖氨酸（K）为谷氨酰胺（Q）时，DdCBE-C41QQ变体的编辑特异性提升；通过三元TALE结构引入Dddi也能够显著降低脱靶编辑。结论：通过不同的结构优化获得了多种DdCBE变体，虽然部分变体会削弱在靶编辑的效率，但都明显降低了脱靶编辑，显著提高了DdCBE的编辑特异性，拓展了线粒体基因组单碱基编辑的工具箱。

An animal model harboring pathogenic mitochondrial DNA (mtDNA) mutations is important to understand the biological links between mtDNA variation and mitochondrial diseases. DdCBE, a DddA-derived cytosine base editor, has been utilized in zebrafish, mice, and rats for tC sequence-context targeting and human mitochondrial disease modeling. However, human pathogenic mtDNA mutations other than the tC context cannot be manipulated. Here, we screened the combination of different DdCBE pairs at pathogenic mtDNA mutation sites with nC (n for a, g, or c) context and identified that the left-G1333C (L1333C) + right G1333N (R1333N) pair could mediate C & BULL;G-to-T & BULL;A conversion effectively at aC sites in rat C6 cells. The editing efficiency at disease-associated mtDNA mutation sites within aC context was further confirmed to be up to 67.89% in vivo. Also, the installed disease-associated mtDNA mutations were germline transmittable. Moreover, the edited rats showed impaired cardiac function and mito-chondrial function, resembling human mitochondrial disease symptoms. In summary, for the first time, we expanded the DdCBE targeting scope to an aC motif and installed the path-ogenic mutation in rats to model human mitochondrial dis-eases.

Hundreds of pathogenic variants of mitochondrial DNA (mtDNA) have been reported to cause mitochondrial diseases, which still lack effective treatments. It is a huge challenge to install these mutations one by one. We repurposed the DddA-derived cytosine base editor to incorporate a premature stop codon in the mtProtein-coding genes to ablate mitochondrial proteins encoded in the mtDNA (mtProteins) instead of installing pathogenic variants and generated a library of both cell and rat resources with mtProtein depletion. In vitro, we depleted 12 of 13 mtProtein-coding genes with high efficiency and specificity, resulting in decreased mtProtein levels and impaired oxidative phosphorylation. Moreover, we generated six conditional knockout rat strains to ablate mtProteins using Cre/loxP system. Mitochondrially encoded ATP synthase membrane subunit 8 and NADH:ubiquinone oxidoreductase core subunit 1 were specifically depleted in heart cells or neurons, resulting in heart failure or abnormal brain development. Our work provides cell and rat resources for studying the function of mtProtein-coding genes and therapeutic strategies.

CRISPR-Cas9技术的发展极大地推动了基因编辑技术的进步,基于该技术开发的碱基编辑器和先导编辑器赋予了CRISPR-Cas9系统更加强大的基因编辑能力,加速了CRISPR-Cas9技术应用于临床基因治疗.虽然目前对该系统做了诸多优化和改进,但在特异性、安全性以及在体转导等方面仍存在改进和优化的空间.本文针对这三方面的研究进展进行简要介绍和展望.